柚皮素激活AMPK诱导急性白血病HL-60细胞内质网应激型自噬和细胞凋亡

Naringin Activates AMPK and Induces ER Stress Autophagy and Apoptosis in HL-60 Cells of Acute Leukemia

目的研究柚皮素(naringenin,NG)抑制H1/0细胞的机制。方法用不同浓度的柚皮素处理HL-60细胞,检测细胞活力;选择无显著细胞毒性的低、中、高3组浓度(5、10、20μmol/L)进行后续实验作为处理组,对照组不做柚皮素处理;Cell Counting Kil(CCK-8)检测HL40细胞增殖倍数;流式细胞术结合蛋白免疫印迹检测HL-60细胞凋亡情况;蛋白免疫印迹结合免疫荧光检测微管相关蛋白轻链3(microtubule-associated protein 1 light chain3,LC3)表达;蛋白免疫印迹检测腺昔酸活化蛋白激酶(adenosine monophosphate activated protein kinase,AMPK)信号通路。结果柚皮素以剂量依赖性的方式抑制HL-60细胞的增殖(P<0.01);流式细胞术结果显示柚皮素以剂量依赖性的方式增加HL-60凋亡细胞比例,柚皮素诱导caspase-3表达增加;免疫荧光结果显示柚皮素诱导细胞核内LC3阳性色斑增加,柚皮素诱导P62表达减少,Beclin1表达增加和LC3-D/LC3-Ⅰ比值增加;柚皮素激活AMPK信号通路。结论柚皮素抑制HL-60细胞的细胞的增殖、促进H1/0细胞凋亡、诱导HL-60细胞内质网应激型自噬,且有可能通过激活AMPK信号通路调控凋亡和自噬,为急性白血病的治疗研究提供新思路和理论基础。

Objective Naringenin (NG), a citnis flavoid, is a widely studied anticancer agent,but its anticancer mechanism is not fully understood. This study investigated the molecular mechanism by which NG stimulates apoptosis and pro-death autopluigy in HL-60 cells of acute leukemia.Methods HL-60 cells were treated with different concentrations of NG and cell viability was detected. 3 groups of the cells were treated with 10 , 25 , 50 μM of NG, and a group of cells without NG treatment served as control group. The cell growth was detected by using CCK-8. The apoptosis rate was flow cytometrically determined. Results NG inhibited the cell growth of HL-60 cells in a dose-dependent manner and the cell growth was reduced in the treatment groups. Cell growth was significantly decreased in the treatment groups as compares with the control group ( P < 0. 01 ).Flow cytometry showed that, after NG treatment, the number of apoptotic cells increased in a dose-dependent fashion and caspase-3 expression was increased. Immunofluorescence showed that NG increased the number of puncta positive for the microtubule-associated protein 1 light chain 3 (LC3), decreased expression of P62 and increased Beclin 1 expression. Moreover, the ratio of LC3-Ⅱ/LC3-Ⅰwas increased. NG activated adenosine monophosphate-activated protein kinase (AMPK) signalling pathways. Conclusion This study suggested that NG inhibited HL-60 proliferation and increased apoptosis and autophagy flux. These activities might be modulated by the activation of AMPK signaling pathways.